Mass spectrometry has become the method of choice for fast and efficient identification of proteins in biological samples. Tandem mass spectrometry of peptides in a complex protein mixture can be used to identify and quantify the proteins present in the original mixture. Tandem mass spectrometers achieve this by selecting single mass-to-charge (m/z) values and subjecting the precursor ions to fragmentation, providing product ions that can be used to sequence and identify peptides. The information created by the product ions of a peptide can be used to search peptide and nucleotide sequence databases to identify the amino acid sequence represented by the spectrum and thus identify the protein—from which the peptide was derived. Analytical methods that compare the fragment ion pattern to theoretical fragment ion patterns generated computationally from sequence databases can be used to identify the peptide sequence. Such methods can identify the best match peptides and statistically determine which peptide sequence is more likely to be correct. The algorithms typically utilize m/z information for identification purposes of the various product ions.
Frequently, tandem mass analysis includes fragmenting a selected precursor (or “parent”) ion and recording the mass spectrum of the resultant fragment ions. The information in the fragment ion mass spectrum is often a useful aid in elucidating the structure of the precursor ion. The general approach used to obtain a tandem mass spectrometry (MS/MS or MS-2) spectrum is to isolate a selected precursor ion with a suitable mass analyzer, and to subject the precursor ion to energetic collisions with a neutral gas so as to analyze the mass of the resulting fragment ions in order to generate a mass spectrum. To obtain more information from a precursor ion, an additional stage of MS can be applied to the MS/MS schemes outlined above, resulting in MS/MS/MS, or MS-3. For example, the collision cell may be operated as an ion trap, wherein fragment ions are resonantly excited to promote further CID.
FIG. 1 depicts a nomenclature typically adopted (and used herein) for the fragments of peptides and proteins. The accepted nomenclature for fragment ions was first proposed by Roepstorff and Fohlman (Roepstorff, P., and J. Fohlman. “Letter to the editors.” Biological Mass Spectrometry 11, no. 11 (1984): 601-601.) and subsequently modified by Johnson et. al. (Johnson, Richard S., Stephen A. Martin, Klaus Biemann, John T. Stults, and J. Throck Watson. “Novel fragmentation process of peptides by collision-induced decomposition in a tandem mass spectrometer: differentiation of leucine and isoleucine.” Analytical chemistry 59, no. 21 (1987): 2621-2625.) both of which are incorporated by reference herein in their entirety. The three possible cleavage points of the peptide backbone are called a, b and c when the charge is retained at the N-terminal fragment of the peptide and x, y and z when the charge is retained by the C-terminal fragment. The numbering indicates, which peptide bond is cleaved counting from the N and the C terminus respectively, and thus also the number of amino acid residues in the fragment ion. The number of hydrogens transferred to or lost from the fragment is indicated with apostrophes to the right and the left of the letter respectively. Peaks corresponding to ions that have lost ammonia (17 Da) and water (18 Da) are denoted, respectively, by superscript asterisks and circles to the right of the letter.
It has been observed that low-energy CID predominantly yields, when fragmentation is along the peptide backbone, fragment ions (ion products) of type a, b, and y; a*, b*, and y* and ao, bo, and yo. By contrast, ETD produces mainly c and z* fragment ions and to a much smaller extent a*, y ions and z′ and c* ions. Conventional analysis techniques are designed to deal with spectra containing only one type of fragments (c/z or b/y).
Targeted MS analysis of peptides is an important analytical procedure. For example, as a therapeutic test, the quantification of peptide abundance can be used as a proxy for the disease state of an organism. Most commonly, targeted MS/MS (MS-2) with liquid chromatography (LC) is used, the conventional method comprising selecting a peptide ion species from the eluting background species with a first stage of mass analysis, fragmenting the selected ion species, and measuring the so-formed fragments. For peptide analysis, the MS-2 experiment is relatively easy to setup, because for a given peptide sequence, the most abundant and selective MS-2 fragments can be predicted with a high probability to be backbone cleavages, where the charge is retained on the peptide C-terminus, or y-ions (see FIG. 1). A typical strategy is to interrogate the y-type ion species that have greater values of m/z than that of the precursor. Although it is usually necessary to validate the presence and selectivity of these transitions, this task is much simpler for a peptide molecule whose fragmentation has not been previously studied than it would be for other molecule classes, because the superset of most probable fragment ion species is known.
Although conventional MS-2 analysis provides a great deal of selectivity and throughput for peptide quantification, it has been estimated by some researchers that an additional 1-2 orders of magnitude of sensitivity are needed to rival the sensitivity levels achieved by immunoassays. One of the means that mass spectrometers have to achieve better sensitivity is to find ways to increase the selectivity of the measurements; that is, to reduce interference that gives rise to a high baseline and overlapping peaks, provided that the signal of the analyte is not reduced as fast as the interference “noise”. For this reason, higher resolution mass analysis is in general preferred over nominal mass resolution, provided that other analytical figures of merit such as speed and signal abundance are sufficient. Other modes of increasing selectivity are increasingly being explored, such as coupling the ion outlet of ion mobility spectrometer to the ion inlet of a mass spectrometer.
Another well-known means of increasing MS selectivity is to perform additional stages of MS beyond MS-2, i.e. further stages of precursor isolation, fragmentation, and fragment-ion measurement. Traditionally, MS-3 analysis has been mostly used for qualitative tasks, such as peptide phospho-peptide site localization (e.g., see Xu, Hua, Liwen Wang, Larry Sallans, and Michael A. Freitas. “A hierarchical MS2/MS3 database search algorithm for automated analysis of phosphopeptide tandem mass spectra.” Proteomics 9, no. 7 (2009): 1763-1770). More recently, MS-3 has been and is being used for global proteome characterization and quantification with isobaric labeling strategies, such as Tandem Mass Tags, in which all MS-2 precursors dissociate to form the same approximately 10 reporter ions in a known mass region (e.g., see McAlister, Graeme C., David P. Nusinow, Mark P. Jedrychowski, Martin War, Edward L. Huttlin, Brian K. Erickson, Ramin Rad, Wilhelm Haas, and Steven P. Gygi. “MultiNotch MS3 enables accurate, sensitive, and multiplexed detection of differential expression across cancer cell line proteomes.” Analytical chemistry 86, no. 14 (2014): 7150-7158). In this method, the activation step to yield reporter ions uses a very high collision energy to drive all products to reporters, and so there is no emphasis on generation of normal peptide backbone diagnostic fragments, which could be used by themselves in a targeted setting.
The use of MS-3 as a general targeted peptide quantification strategy has thus not been well explored up to this point. The main reason is that, although the potential selectivity payoff is large, MS-3 has traditionally lacked sensitivity, so that very large dwell times have been needed to generate enough fragment ions to be analytically useful. For example, Lemoine et al (Lemoine, Jérôme, Tanguy Fortin, Arnaud Salvador, Aurore Jaffuel, Jean-Philippe Charrier, and Genevieve Choquet-Kastylevsky. “The current status of clinical proteomics and the use of MRM and MRM3 for biomarker validation.” Expert review of molecular diagnostics 12, no. 4 (2012): 333-342) report that typical periods between targeted MS-3 scans were 300 ms. This problem has been ameliorated to a considerable extent with the advent of multinotch isolation (op. cit.), where multiple MS-2 fragments are simultaneously isolated and fragmented, such that the period between scans can be on the order of 30 ms. Nonetheless, there remains a need for a general-purpose procedure for performing multiplexed MS-3 on any particular given peptide sequence, especially in the absence of prior knowledge about the peptide's fragmentation behavior. This is important, because a typical translational workflow between discovery and targeted proteomics could involve the analysis of hundreds or thousands of peptides, leaving little time for the manual optimization of parameters for several different peptides of interest or of potential interest. The presently-known MS-3 methods would require a priori information regarding which MS-2 fragment ions should be isolated and further fragmented, which MS-3 fragment ions are formed from the activation of the MS-2 fragment ions and which of these are most useful for quantitation. The methods of the present teachings address the above-noted need in the art by advantageously providing the ability to make or obtain qualitative and quantitative analysis of certain peptide analytes, even in situations in which the precise fragmentation behavior of the analyte or analytes is not known in advance.